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useful for screening of natural extracts and synthetic chemicals for potential nuclear receptor ligands.

Receptor Cofactor Assay System [RCAS] kit for Estrogen receptor alpha (ER)

Background

Estrogen receptor alpha (ERa) is a member of the nuclear receptor (NR) family. NR is one of the transcription factors that regulate target gene expression. ERa plays an important role of reproduction in physiological functions, therefore, the study of chemical binding to ERa is very useful for research of endocrine disrupting chemicals (EDCs) and drug screening.

NR activation before gene expression consists of 3 steps.

 Step1: Ligand binding to NR induces conformational change of NR

 Step2: Coactivator is recruited to NR-ligand complex

 Step3: Other transcriptional factors are recruited to NR-ligand-coactivator complex

Competitive binding assay detects only the ligand-binding to NR, whereas RCAS detects NR-ligand-coactivator complex. Therefore you can predict the characteristic of your test samples more accurately using this RCAS kit (i.e. both agonist and antagonist for ERa can be detected.).

Principle

A peptide containing LXXLL motif of coactivator (SRC1) is immobilized on the microwell plate. The mixture of recombinant human ERa with potentially agonistic or antagonistic compounds is incubated into the plate. The binding of ERa-ligand complex to the coactivator peptide on the plate is detected by using HRP conjugated detection antibody.
The HRP activity is determined by the addition of TMB substrate solution. The reaction is stopped by addition of an acid solution and the resultant color read at 450 nm using a microwell plate spectrophotometer. The reactivity of the sample to the receptor can be determined by calculation of EC50 or IC50 using the absorbance data.



A typical result

1) Dose-response curve of 17β-estradiol (E2) based on raw optical density data obtained from a 5-fold dilution series of E2 from 40 nM.

 

2) Actual Photograph of TMB color development in the wells just before adding the stop solution (the photograph was taken when the data described in panel A was measured.)

 

2) Example of data analysis using SRC1 (+) and SRC1 (-)
Panel A shows OD values of the samples observed in SRC1 (+) and SRC1 (-).  Panel B indicates the difference in the observed OD values of each sample [SRC1 (+) SRC1 (-)]. The observed OD values of Sample A and Sample B are almost the same in SRC (+), but because of higher OD value of Sample B in SRC1 (-), Sample A turns out to be more active.

 

4) Typical assay data for agonist (E2, DES, Genistein, NP, BPA and Resveratrol) and antagonist (4-Hydroxytamoxifen and ICI182,780)
The EC50 or IC50 value of test chemicals will be calculated by logistic 4-parameter curve fit using computer software such as Prism 4 (GraphPad).


Dose response curve of 17beta-estradiol (E2), Diethylstilbestrol (DES), Genistein, 4-Nonylphenol (NP), Bisphenol A (BPA) and Resveratrol.


Dose response curve of 17beta-estradiol (E2), 4-Hydroxytamoxifen (antagonist, in combination with 2 nM E2) and ICI182,780 (antagonist, in combination with 2 nM E2)

 

Product List

Product Name Cat# Quantity Price

EnBio RCAS for ER (ALPHA)

EBT-ERA-SRC-EX 1SET

¥ 100,000
$ 1334
€ 1000

EnBio RCAS for PPAR(DELTA)

EBT-PPARD-CBP-EX 1SET

¥ 100,000
$ 1334
€ 1000

EnBio RCAS for PPAR(GAMMA)

EBT-PPARG-CBP-EX 1SET

¥ 100,000
$ 1334
€ 1000

EnBio RCAS for PPAR(ALPHA)

EBT-PPARA-CBP-EX 1SET

¥ 100,000
$ 1334
€ 1000

EnBio RCAS for LXR(ALPHA)

EBT-LXRA-SRC-EX 1SET

¥ 100,000
$ 1334
€ 1000

The product “EnBio RCAS for ER β Cat#EBT-ERB-SRC-EX” is not sold to the following countries by the patent issue.. 

United States, Canada, Australia, Austria, Belgium, Bulgaria, Switzerland, Cyprus, Czech Republic, Germany, Denmark, Estonia, Spain, Finland, France, England, Greece, Hungary, Ireland, Iceland, Italy, Liechtenstein, Lithuania, Luxembourg, Latvia, Monaco, Netherlands, Poland, Portugal, Romania, Sweden, Slovene, Slovakia, and Turkey

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